1. Field of the Invention
The present invention relates to the field of analytical test elements and methods, such as are useful in manual and automated diagnostic systems, and, more particularly, to multilayer analytical elements useful in the qualitative and quantitative determination of body fluid constituents and medicaments present in such body fluids.
2. Brief Description of the Prior Art
Test devices in the form of test strips and similar solid state analytical elements have become commonplace in the analysis of various types of samples, particularly biological fluids. Test strips designed for detecting clinically significant substances in biological fluids, such as serum and urine, have been advantageous in the diagnosis of disease.
Test strips of various types have been known and used for many years in a wide variety of fields, from the most familiar pH test paper devices to in vitro dignostic devices for the detection of various urine and blood components such as glucose, protein, occult blood and so forth (e.g., as described in U.S. Pat. Nos. 3,164,534; 3,485,587; and 3,012,976). Reagent compositions found in such test strips, often having limited sensitivity, interact with the constituent or constituents to be determined by direct chemical reaction and are applied to the detection of substances that are present in liquid samples at concentrations in the millimolar range or above.
(a) Multilayer Analytical Elements
A basic multilayer integral analytical element is described in U.S. Pat. No. 3,092,465. Such multilayer elements use an absorbent firbrous carrier impregnated with one or more reagents, typically including a color former, over which is coated a semi-permeable membrane. Upon contact with a test liquid, analyte passes through the membrane and into the fibrous carrier to generate color in an amount related to the concentration of analyte. The membrane prevents passage and absorption of certain interfering components such as red blood cells, that could impair accurate reading of the color provided as a test result.
Other multilayer integral analytical elements are described in U.S. Pat. No. 3,992,158. Such elements can receive a liquid sample and spread the sample within a spreading layer of the element to obtain in the element an apparent uniform concentration of analyte, other appropriate sample constituent or analyte product and produce in the presence of analyte an analytical result that can be measured quantitatively by automated devices, using techniques such as spectrophotometry, fluorimetry, etc. Such elements can include spreading layers and reagent layers that contain a reactive or otherwise interactive material that, by virture of its activity, promotes in the element a radiometrically detectable change, such as a color change.
U.S. Pat. No. 4,042,355 relates to an element having (1) a reagent layer which reacts with the analyte to form a diffusible, detectable species; (2) a nonfibrous radiation blocking layer, permeable to the detectable species and having an opacifying agent; and (3) a nonfibrous, radiation-transmissive registration layer in which the detectable species is detected. The element is, thus, read from below.
U.S. Pat. No. 4,066,403 (Re 30,267) relates to an element including () a reagent which reacts with the analyte to produce a decomposition product; and (2) a reagent which reacts with the decomposition product or an intermediate to provide a detectable change, and having, as an improvement, a barrier composition separating reagent (1) from reagent (2), and being substantially uniformly permeable to the decomposition product and substantially impermeable to interferants. Therefore, what this does is add a "filtering" layer between the "reagent" layer and the "registration" layer.
U.S. Pat. No. 4,144,306 relates to an element in which the reagent layer contains a nondiffusible material including a preformed, detectable moiety which is released and becomes diffusible in the presence of the analyte. The registration layer receives the diffusible species. Layers within the element are composed such that the preformed, detectable moiety released from the reagent layer can be detected selectively within the element.
U.S. Pat. No. 4,166,093 relates to an element having (1) a radiation-transmissive reagent layer that reacts with an analyte to provide a detectable species, and (2) a porous radiation-blocking layer which is permeable to the analyte. As an improvement, it also has (3) a radiation-transmissive, detectable species migration-inhibiting layer between the reagent layer and the porous radiation-blocking layer. The migration-inhibiting layer is permeable to the analyte and inhibits the migration of the detectable species to the radiation-blocking layer.
(b) Specific Binding Assay Device
Solid phase test devices have been applied to heterogeneous specific binding assays in attempts to overcome the inconveniences and disadvantages of the requisite separation step. A commonly used solid phase device of this type comprises a nonporous surface, such as the interior surface of a test tube or other vessel, to which antibody is affixed or coated by adsorption or covalent coupling. U.S. Pat. Nos. 3,826,619; 4,001,583; 4,017,597; and 4,105,410 relate to the use of antibody coated test tubes in radioimmunoassays. Solid phase test devices have also been used in heterogeneous enzyme immunoassays (U.S. Pat. Nos. 4,016,043 and 4,147,752) and in heterogeneous fluorescent immunoassays (U.S. Pat. Nos. 4,025,310 and 4,056,724; and British Pat. Spec. No. 1,552,374).
The use of such heterogeneous specific binding assay test devices is exemplified by the method of U.S. Pat. No. 4,135,884 relating to a so-called "gamma stick". The test device is incorporated with the antibody reagent and is brought into contact with the liquid sample and with remaining reagents of the reaction system, principally the label conjugate. After an incubation period, the solid phase device is physically removed from the reaction solution and the label is measured either in the solution or on the test device.
Similar devices where the antibody reagent is entrapped in a matrix such as a gel or paper web are described in U.S. Pat. Nos. 3,925,017; 3,970,429; 4,138,474; 3,966,897; 3,981,981 and 3,888,629 and in German OLS No. 2,241,646. Likewise, devices for use in heterogeneous specific binding assays wherein the antibody reagent is fixed to a matrix held in a flowthrough column are known. (U.S. Pat. Nos. 4,036,947; 4,039,652; 4,059,684; 4,153,675; and 4,166,102). The test device is usually incorporated with less than all of the necessary reagents for carrying out the assay and is merely a means for rendering the necessary separation step more convenient.
Finally, heterogeneous specific binding assay test devices have been described wherein most or all of the necessary reagents are incorporated with the same carrier element, and wherein reagent/sample contacts and separation of the free- and bound-phases are accomplished by capillary migrations along the carrier element (U.S. Pat. Nos. 3,641,235; 4,094,647 and 4,168,146). The devices described in such patents are generally considered difficult to manufacture and susceptible to irreproducibility due to the complex nature of the many chemical and physical interactions that take place along the carrier element during performance of an assay. Yet another approach to a heterogeneous immunoassay element is exemplified by U.S. Ser. No. 973,669, published as European Patent Application 0 013 156.
The application of homogeneous specific binding assay reagent systems to solid state test devices would provide great advantages to the routine user of such assay systems. The determination of ligands appearing in very low concentrations in liquid samples would be simplified to the steps of contacting the device with the sample and measuring, either by visual observation or by instrumental means, the resulting signal. Reagents would be provided in a solid form, with no need to store, dispense or mix liquid reagents as required when using the prior art test kits. Solid state devices would also be much more adaptable to automation than the prior art liquid systems.
British Patent Spec. No. 1,552,607, commonly assigned herewith, describes homogeneous specific binding assay systems employing various novel labels, including chemiluminescent labels, enzyme substrate labels and coenzyme labels. At page 23, line 12 et seq of this patent there is the suggestion to incorporate the assay reagents with various carriers including liquid-holding vessels or insoluble, porous, and preferably absorbent, matrices, fleeces, or flocks; gels; and the like. This lacks a detailed teaching of how to apply homogeneous specific binding assay reagent systems to solid state test devices.
German OLS No. 2,537,275 describes a homogeneous specific binding assay reagent system and poses the possibility of using slides or strips incorporated with antibody in performing the assay. In this suggestion, the label conjugate would first be mixed with the sample and thereafter the antibody incorporated test device contacted with the reaction mixture. After a suitable incubation time, it is proposed that the test device would be rinsed with buffer, dried, and then the signal (fluorescence) measured. Thus, this German OLS poses a test device and assay method much like those already known for heterogeneous specific binding assay techniques wherein the test device is immersed in the liquid reaction mixture, incubated, thereafter removed, washed, and finally read. Additionally, the proposed test device does not incorporate all of the binding assay reagents with the carrier element. Specifically, only the antibody is proposed to be incorporated with the carrier element, the label conjugate being separately added to the sample under assay prior to contact with the proposed test device.
Copending U.S. Ser. No. 255,521, filed on Apr. 20, 1981, and commonly assigned herewith, discloses a method for determining the presence of a ligand in or the ligand binding capacity of a liquid test sample, the method comprising the steps of (1 ) adding to the liquid sample a label conjugate comprising the ligand, or a binding analogue thereof, chemically bound to a label, (2) contacting the sample with a test device comprising a carrier matrix incorporated with reagents which, when combined with the label conjugate, produce a homogeneous specific binding assay system which produces a detectable response which is a function of the presence of the ligand or the ligand binding capacity, thereby producing the response, and (3) measuring the response.
Copending U.S. Ser. No. 202,378, filed on Oct. 30,1980, now abandoned, and commonly assigned herewith, discloses a homogeneous specific binding assay device, a method for its preparation, and a method for its use in determining a ligand in or the ligand binding capacity of a liquid sample. This includes, for example, a test device for determining a ligand in or the ligand binding capacity of a liquid sample, comprising (a) reagents for a homogeneous specific binding assay system which produces a detectable response that is a function of the presence of the ligand in or the ligand binding capacity of the sample, and (b) a solid carrier member incorporated with the reagents.
Copending U.S. Ser. No. 253,147, filed on Apr. 10, 1981, and commonly assigned herewith, discloses a homogeneous specific binding assay device for use in determining a ligand in a liquid sample, comprising (a) a reagent composition including a complex of (i) a label conjugate comprising a label component coupled to the ligand or a specific binding analog thereof, and (ii) a specific binding partner for the ligand, the label providing a detectable response, or interacting with a detectant system to provide a detectable response, which is different when the label conjugate is bound by the binding partner compared to when it is not so bound and (b) a carrier incorporated with the complex.